Recent advances in knowledge of the structure/function relationships of LAGLIDADG homing endonucleases (LHE's) has allowed the creation of artificial LHE's with novel recognition and cut sites, suggesting the possibility that artificial LHE's can be engineered to cut at specific target sites in the human genome as a means to enhance homologous recombination at those sites. In this grant, we propose to develop and evaluate methods for introducing LHE's and DNA repair templates into hematopoietic cells for the purpose of targeted gene repair of primary immune deficiencies. We have chosen as our initial target the IL2Rgamma gene, involved in X-linked severe combined immune deficiency syndrome (XSCID), as it is the most common form of severe combined immune deficiency (SCID), and there is evidence for the existence of a selective advantage for cells expressing a functional version of the protein encoded by XSCID locus. The latter property is of great importance, as it allows future in-vivo attempts at gene repair which are successful in only a few percent of a target cell population to be amplified to a level sufficient to make a clinically relevant impact on immune function. In Specific Aim 1, we will construct models for benchmarking LHE-induced targeted repair of the IL2Rgamma gene. Subaim a will involve engineering cultured lymphocyte cell lines to carry cut sites for I-Scel and I-Anil LHEs near or within exon 6 of the IL2Rgamma gene. We will then use these cell lines in subaim b to evaluate various methods for introducing the I-Scel or I-Ani LHE's and a DNA repair template as a means to induce targeted repair of the IL2Rgamma gene in a hematopoietic/lymphocytic cell context. In Specific Aim 2. we will modify the IAni LHE to cut at specific sites we have identified, based on principles for modification of LHE's defined by our collaborators, at or near exon 6 of the human or murine IL2Rgamma genes. In subaim a, we will use a combination of genetic screens, biochemical evaluation, and rationale design to identify I-Anil variants which cut at the identified sequences. In subaim b, we will use the models and methods developed in specific Aim 1 to assay each modified LHE for its capacity to induce targeted gene repair as well as its effects on genomic integrity, with I-Scel-induced targeted repair serving as a comparison benchmark. Overall, the work proposed here will build a foundation of materials and experience for the development of LHE's as general tools for targeted gene therapy of human primary immune deficiencies.